Stock Solutions

Make up 50% w/v with H20 and filter-sterilize with a 0.45uM filter unit (Nalgene). We don't autoclave the PEG. Store in a tightly capped container to avoid evaporation.

Single-stranded carrier DNA (Sigma D1626)

Weight out 200mg of the DNA into 100ml of TE buffer. Disperse the Dna into solution by drawing it p and dwn repeatedly in a 10-ml pipette. Mix vigorosly on a magnetic stirrer for 2-3 hours or until fully dissolved. Alternatively, leave the covered solution mixing at this stage overnight in a cold rom.

Aliquot the DNA into 100μL portions and store at -20°C.

Prior to use, the aliquot should be boiled and then quickly cooled on ice. We use a thermocycler to heat the DNA to 95°C for 25 minutes and then rapidly cool it on ice.

Once the salmon sperm has been boiled it can be freeze-thawed 3 or 4 times before transformation efficiencies begin to decrease. In practice, we boil the DNA before every transformation.

TE Buffer (pH 8.0)

10 mM Tris-HCL (pH 8.0)

1.0 mM EDTA

1.0M Lithium acetate stock solution (LiAc)

Prepare as a 1.0 M stock in distilled deionized H2O; filter-sterilize. The final pH should be between 8.4 and 8.9

Day 1

Inoculate the strain to transform from a single colony into 5mls of YPD in a test tube. Put on the roller drum at 30°C overnight.

Day 2

Notes

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